Crystallization and refinement

For K_2P2.1_cryst ML336 crystals, ML336 was dissolved in 100% DMSO at a concentration of 500 mM, diluted 1:100 in SEC buffer to 5 mM concentration, and then mixed to dissolve the compound completely. 12 mg ml⁻¹ K_2P2.1_cryst then mixed 1:1 with the ML336-SEC buffer solution to achieve a final concentration of 6 mg ml⁻¹ and 2.5mM of K_2P2.1_cryst and ML336, respectively. This mixture was then incubated for 2h on ice prior to crystallization using hanging-drop vapor diffusion at 4 °C and mixture of 0.2 μl of protein and 0.1 μl of precipitant over 100 μl of reservoir containing 20–25% PEG400, 200 mM KCl, 100 mM HEPES pH 8.0, 1 mM CdCl₂. Crystals appeared in 12 h and grew to full size (200–300 μM) in about a week. Crystals were cryoprotected with buffer D (200 mM KCl, 0.2% OGNG, 15 mM HTG, 0.02% CHS, 100 mM HEPES pH 8.0,1 mM CdCl₂) with 5% step increase of PEG400 up to a final concentration of 38% and flash-frozen in liquid nitrogen.

TREK-1^CG*:CAT335 crystals, TREK-1^CG*:CAT335a crystals, and TREK-1^CG*:ML335 crystals grew in the similar conditions as the TREK-1:ML336 complex, but CAT335 and CAT335a were dissolved in 100% DMSO at a concentration of 20 mM and ML335 was dissolved in 100% DMSO at a concentration of 500 mM. Each ligand was diluted in SEC buffer and mixed 1:1 volume ratio to TREK-1^CG* previously concentrated to 10 mg ml⁻¹ to achieve final ligand:protein molar ratios of 1.1:1 CAT335, 2:1 CAT335a, and 5:1 ML335. These mixtures were incubated for 2h on ice prior to crystallization using hanging-drop diffusion at 4 °C and a mixture of 0.2 μl of protein and 0.1 μl of precipitant over 100 μl of reservoir containing 20–25% PEG400, 200 mM KCl, 100 mM HEPES pH 7.0 or 7.5, and 1 mM CdCl₂. Crystals appeared in 12 h and grew to full size (200–300 μM) in about a week. Crystals were cryoprotected with buffer D (200 mM KCl, 0.2% OGNG, 15 mM HTG, 0.02% CHS, 100 mM HEPES pH 7.0 or 7.5, and 1 mM CdCl₂) with 5% step increase of PEG400 up to a final concentration of 38% and flash-frozen in liquid nitrogen.

Datasets for K_2P2.1–ligand complexes were collected at 100 K using synchrotron radiation at APS GM/CAT beamline 23-IDB/B Chicago, Illinois using a wavelength of 0.9779 Å, processed with XDS⁷⁵, scaled and merged with Aimless⁷⁶. Final resolution cut-off was 2.9 Å (K_2P2.1_cryst:ML336) and 3.0 Å (K_2P2.1^CG*:CAT335, K_2P2.1^CG*:CAT335a, and K_2P2.1^CG*:ML335), using the CC_1/2 criterion^77,78. Structures were solved by molecular replacement using the K_2P2.1_cryst structure (PDB: 6CQ6) as search model. Several cycles of manual rebuilding, using COOT⁷⁹ and refinement using REFMAC5⁸⁰ and PHENIX⁸¹ were carried out to improve the electron density map. Ramachandran restraints were employed during refinement. Model quality was assessed using Molprobity⁸².